Recent years have seen increased effort directed toward development of protein-based anti-tumor drugs. Thus, a number of monoclonal antibodies have been approved by the FDA for the treatment of various cancers. A related and potentially more powerful approach which makes use of the high specificity binding of antibodies to tumor markers is the development of immunotoxins, fusions of tumor-specific antibodies to bacterial or plant toxins. In this laboratory, efforts have emphasized an alternative approach to achieving tumor cell specificity by using modified anthrax toxin. Anthrax toxin depends on proteolytic activation of the receptor-bound protective antigen protein (PA) by cell surface proteases. Replacing the furin-cleaved sequence with sequences recognized by matrix metalloproteases or urokinase plasminogen activator has yielded tumor-specific anti-cancer fusion proteins that are efficacious in mouse tumor models. Both the wildtype and the modified PA assemble into oligomeric protein-conducting channels that delivers the anthrax toxin catalytic effector proteins lethal factor (LF) and edema factor (RF) to endosomes and then translocates them to the cytosol. For targeting of tumors, the native anthrax effector protein LF was replaced with a fusion containing the N-terminal PA-binding 254 amino acid-containing domain of anthrax toxin lethal factor (LFn) and the Pseudomonas aeruginosa exotoxin A (PE) catalytic domain (PEIII) to obtain the fusion protein FP59. The LFn domain delivers PEIII to the cytosol and PEIII transfers ADP-ribose to eukaryotic elongation factor 2 (eEF2), resulting in protein synthesis inhibition and cell death. This system is highly effective in achieving tumor-specific cell-surface PA activation and cytosolic delivery of PEIII. It has been successfully tested for a number of tumor types, and is expected to be active on nearly all types of solid tumors. The cytosolic activity of effector molecules like PEIII depends greatly on their avoidance of cytosolic degradation, e.g. by the proteasome. Proteins targeted for degradation are labeled by ubiquitin and directed to the 26S proteasome. Alexander Varshavsky identified the N-terminal amino acid of certain proteins as controlling their cytosolic stability. This N-end rule identified certain amino acids at the N-terminus to be stabilizing residues (e.g. Met, Gly, or Ala) while other residues clearly result in faster protein degradation (e.g. Arg, Lys, or Asp). We previously showed that certain stabilizing N-terminal amino acids increased stability of LF and FP59 approximately 5-fold in the cell cytosol and increased cytotoxicity toward both cells and animals. During 2014 we extended this work to EF, which was known to have relatively short-lived action on cells. EF is a calmodulin-dependent adenylate cyclase that converts adenosine triphosphate (ATP) into 35-cyclic adenosine monophosphate (cAMP), contributing to the establishment of B. anthracis infections and the resulting pathophysiology. We showed that EF adenylate cyclase toxin activity is strongly mediated by the N-end rule, and thus is dependent on the identity of the N-terminal amino acid. EF variants having different N-terminal residues varied by more than 100-fold in potency toward cultured cells and mice. EF variants having unfavorable, destabilizing N-terminal residues showed much greater activity in cells when the E1 ubiquitin ligase was inactivated or when proteasome inhibitors were present. Taken together, these results show that EF is uniquely affected by ubiquitination and/or proteasomal degradation. We characterized an anti-cancer fusion protein consisting of anthrax lethal factor (LF) and the catalytic domain of Pseudomonas exotoxin A by (i) mutating the N-terminal amino acids and by (ii) reductive methylation to dimethylate all lysines. Dimethylation of lysines was achieved quantitatively and specifically without affecting binding of the fusion protein to PA or decreasing the enzymatic activity of the catalytic moiety. Ubiquitination in vitro was drastically decreased for both the N-terminally mutated and dimethylated variants, and both appeared to be slightly more stable in the cytosol of treated cells. The dimethylated variant showed greatly reduced neutralization by antibodies to LF. The two described modifications offer unique advantages such as increased cytotoxic activity and diminished antibody recognition, and thus may be applicable to other therapeutic proteins that act in the cytosol of cells. Also during 2014, we used our experience in working with multi-component bacterial toxins to study the properties of the Hbl and Nhe hemolysins of Bacillus cereus. This bacteria is a spore-forming, Gram-positive bacterium commonly associated with outbreaks of food poisoning. It is also known as an opportunistic pathogen causing clinical infections such as bacteremia, meningitis, pneumonia, and gas gangrene-like cutaneous infections, mostly in immunocompromised patients. B. cereus secretes a plethora of toxins of which four are associated with the symptoms of food poisoning. Two of these, the (putatively) non-hemolytic enterotoxin Nhe and the hemolysin BL (Hbl) toxin, are predicted to be structurally similar and are unique in that they require the combined action of three toxin proteins to induce cell lysis. Despite their dominant role in disease, the molecular mechanism of their toxic function is still poorly understood. We found that B. cereus strain ATCC 10876 harbors not only genes encoding Nhe, but also two copies of the hbl genes. We identified Hbl as the major secreted toxin responsible for inducing rapid cell lysis both in cultured cells and in an intraperitoneal mouse toxicity model. Antibody neutralization and deletion of Hbl-encoding genes resulted in significant reductions of cytotoxic activity. We purified large amounts of each of the six Hbl and Nhe proteins and characterized their interaction with cells. Microscopy studies with Chinese Hamster Ovary cells showed that pore formation by both Hbl and Nhe occurs through a stepwise, sequential binding of toxin components to the cell surface and to each other. This begins with binding of Hbl-B or NheC to the eukaryotic membrane, and is followed by the recruitment of Hbl-L1 or NheB, respectively, followed by the corresponding third protein. Lastly, toxin component complementation studies indicate that although Hbl and Nhe can be expressed simultaneously and are predicted to be structurally similar, they are incompatible and cannot complement each other. The availability of the purified proteins provides opportunities to analyze their structures, mode of interaction, and specificity for cell binding and membrane permeabilization.